Chondroitinases are enzymes of bacterial origin that act on chondroitin sulfate, a component of the proteoglycans that mediate the attachment between the retina and the vitreous body of the human eye. Examples of chondroitinase enzymes are chondroitinase ABC which is produced by the bacterium Proteus vulgaris (P. vulgaris), and chondroitinase AC, which is produced by A. aurescens. Chondroitinase ABC is designated as chondroitinase I in the present invention. Chondroitinases ABC and AC function by degrading polysaccharide side chains in protein-polysaccharide complexes, without degrading the protein core.
When resolved by SDS-PAGE, chondroitinase I migrates with an apparent molecular mass of about 110 kDa (see FIG. 1). Others have reported the appearance of a doublet in SDS-PAGE resolution of chondroitinase I (Sato et al., Agric. Biol. Chem. 50:4,1057-1059 (1986)). The present inventors have discovered that this doublet represents intact chondroitinase I and 90 a kDa degradation product. Commercial chondroitinase I protein preparations contain variable amounts of 90 kDa protein and 18 kDa protein degradation product fragments (see FIG. 1).
Yamagata et al. (J. Biol. Chem. 243:1523-1535 (1968)) describe the purification of the chondroitinase ABC from extracts of P. vulgaris. This enzyme selectively degrades the glycosaminoglycans chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate (also referred to respectively as chondroitin sulfates A, B, and C which are side chains of proteoglycans) at pH 8 at higher rates than chondroitin or hyaluronic acid. The products of the degradation are large molecular weight unsaturated oligosaccharides and an unsaturated disaccharide. However, chondroitinase ABC does not attack keratosulfate, heparin or heparitin sulfate.
Kikuchi et al., U.S. Pat. No. 5,198,355, describe the purification of glycosaminoglyean degrading enzymes, such as chondroitinase ABC, by fractionating the enzyme by adsorbing a solution containing the enzyme onto an insoluble sulfated polysaccharide carrier and then desorbing the individual enzyme from the carrier.
The chondroitinase enzymes have use in ocular surgery as a means for rapid and specific non-surgical disruption of the attachment of the vitreous body to the neural retina of the eye, thereby facilitating removal of the vitreous body. For example, Hageman, U.S. Pat. No. 5,292,509, describes an ophthalmic vitrectomy method for selectively and completely disinserting (removing) the ocular vitreous body, epiretinal membranes, or fibrocellular membranes from the neural retina, ciliary epithelium and posterior lens surface of the mammalian eye as an adjunct to vitrectomy. An effective amount of an enzyme which disrupts or degrades chondroitin sulfate proteoglycan localized specifically to sites of vitreoretinal adhesion is administered to the eye thereby permitting complete disinsertion of the vitreous body and/or epiretinal membranes. The enzyme can be a protease-free glycosaminoglycanase. Hageman utilized chondroitinase ABC obtained from Seikagaku Kogyo Co., Ltd., Tokyo, Japan, an impure preparation that typically includes chondroitinase I degradation products and/or proteinaceous stabilizers. Proteinaceous stabilizers are undesirable because they are unacceptable in therapeutic or surgical compositions to be administered to the human eye.
Some chondroitinases have also been described as having value in dissolving the cartilage of herniated discs without disturbing the stabilizing collagen components of discs.
Brown, U.S. Pat. No. 4,696,816, describes a method for treating intervertebral disc displacement in mammals, including humans, by injecting into the intervertebral disc space effective amounts of a solution containing chondroitinase ABC. The chondroitinase ABC was isolated and purified from extracts of P. vulgaris. This native enzyme material functioned to dissolve cartilage, such as herniated spinal discs. Specifically, the enzyme causes the selective chemonucleolysis of the nucleus pulposus which contains proteoglycans and randomly dispersed collagen fibers.
Another chondroitinase, chondroitinase II, has now been isolated and purified from P. vulgaris by the present inventors. Additionally, the present inventors have discovered an efficient copurification method in which a mixture chondroitinase I and chondroitinase II can be conveniently obtained.